Increase in candidemia cases and emergence of fluconazole-resistant Candida parapsilosis and C. auris isolates in a tertiary care academic hospital during the COVID-19 pandemic, Greece, 2020 to 2023

Background The COVID-19 pandemic and the emergence of Candida auris have changed the epidemiological landscape of candidaemia worldwide. Aim We compared the epidemiological trends of candidaemia in a Greek tertiary academic hospital before (2009–2018) and during the early COVID-19 (2020–2021) and late COVID-19/early post-pandemic (2022–2023) era. Methods Incidence rates, species distribution, antifungal susceptibility profile and antifungal consumption were recorded, and one-way ANOVA or Fisher’s exact test performed. Species were identified by MALDI-ToF MS, and in vitro susceptibility determined with CLSI M27-Ed4 for C. auris and the EUCAST-E.DEF 7.3.2 for other Candida spp. Results In total, 370 candidaemia episodes were recorded during the COVID-19 pandemic. Infection incidence (2.0 episodes/10,000 hospital bed days before, 3.9 during the early and 5.1 during the late COVID-19 era, p < 0.0001), C. auris (0%, 9% and 33%, p < 0.0001) and fluconazole-resistant C. parapsilosis species complex (SC) (20%, 24% and 33%, p = 0.06) infections increased over time, with the latter not associated with increase in fluconazole/voriconazole consumption. A significant increase over time was observed in fluconazole-resistant isolates regardless of species (8%, 17% and 41%, p < 0.0001). Resistance to amphotericin B or echinocandins was not recorded, with the exception of a single pan-echinocandin-resistant C. auris strain. Conclusion Candidaemia incidence nearly tripled during the COVID-19 era, with C. auris among the major causative agents and increasing fluconazole resistance in C. parapsilosis SC. Almost half of Candida isolates were fluconazole-resistant, underscoring the need for increased awareness and strict implementation of infection control measures.


Introduction
Since the onset of the COVID-19 pandemic, a growing number of studies have indicated a significant rise in the number of secondary invasive fungal diseases, including candidaemia [1].In fact, the global spread of the COVID-19 outbreak posed serious healthcare challenges, comprising staff and equipment shortages, as well as collateral damage to infection control measures [2].More recently, the exponential increase and geographical spread of Candida auris-related invasive infections have raised concerns [3,4].The epidemiology of C. auris bloodstream infections (BSI) in Greece has been described only in a limited number of cases [4,5], mainly in COVID-19 critically ill patients [6][7][8], which may not be indicative of the extent of their occurrence in the general patient population of Greece.
Meanwhile, although guidelines exist to guide therapeutic decisions [9,10], management of candidaemic patients may require a tailored approach owing to the considerable geographical variation in the distribution of the causative pathogens and their corresponding resistance patterns [11].Hence, empirical antifungal strategies must be guided by local epidemiology, taking into account the local ecology as determined through diligent surveillance with provision of feedback at regular intervals [12].To date, the contemporary epidemiology of candidaemia in Greece remains unknown since the little data available focused mostly on intensive care unit (ICU)-acquired infections [7,8,13,14].
In March 2020, Attikon University General Hospital was appointed as one of the referral centres providing COVID-19 care services in Attica, which is the most densely populated region of Greece and encompasses the entire metropolitan area of Athens.During the first 2 years of the pandemic, several changes in Attikon's routine workflow were implemented in order to accommodate the surge of COVID-19 patients.These included the cancellation of non-urgent surgical and medical procedures as well as outpatient visits, emergency conversion of one of the two existing ICUs to an exclusively COVID-19 ICU and the creation of a new ICU, and transformation of several wards to specialised COVID-19 non-critical care units that were functioning for various time periods depending on the gravity of the transmission waves of COVID-19 in Greece.Of note, Attikon is a tertiary academic hospital that attends cases of high complexity, including adult, paediatric and neonatal ICUs, haematology and oncology wards as well as bone marrow transplantation and HIV/AIDS units, whose services could not be completely interrupted, but were diminished to the least possible degree.
We performed a systematic review of the existing literature related to candidaemia in Greece during the COVID-19 pandemic and investigated the epidemiology of the infection before [15] and during the pandemic up to December 2023 in Attikon to provide an overview of the epidemiological situation and the impact of the pandemic.

Literature review
We carried out electronic searches in the PubMed, Google Scholar and Web of Science databases using the keywords 'Candida', 'fungaemia', 'candidaemia' and 'bloodstream infection' in conjunction with 'Greece' and/or 'Greek' in February 2024.Additional handpicked searches in the bibliographies of the articles retrieved were also performed.Only studies written in English and conducted during the COVID-19 era (1 January 2020 to 31 December 2023), were considered.From the studies retrieved, we extracted data regarding the geographic region, observation time period, study design, patient population, number of cases, candidaemia incidence rates, the relative proportions of Candida spp.and the antifungal susceptibility profiles of isolates (including the methodology and interpretation criteria used).

Study period, setting and population
All microbiologically confirmed candidaemia cases in adult patients hospitalised at Attikon between (i) 2009 and 2018 (pre-COVID-19 era) [15], (ii) 1 January 2020 and 31 December 2021 (early COVID-19 era), and (iii) 1 January 2022 and 31 December 2023 (late COVID-19/ early post-pandemic era, hereafter referred to as late COVID-19 era), were retrospectively analysed.Attikon is a modern teaching hospital and the largest in the West Attica region (western part of the agglomeration of Athens), with a potential of 750 beds and an average of 52,220 admissions per year in the pre-COVID-19 era [15].It serves West and South Attica as well as most Aegean islands (second regional health authority of a population of ca. 2 million people).
Candidaemia was defined as the recovery of Candida spp.from at least one blood culture obtained during hospitalisation.Subsequent positive blood cultures with the same Candida spp.from a single patient were considered a new episode if the episodes occurred more than 4 weeks apart, along with the clearance of the prior blood culture (negative blood culture 14 days after the first negative blood culture) and resolution of all clinical features of the infection.Blood cultures yielding different Candida spp., independently of the time interval between the new

Table 1a
Epidemiological aspects of candidaemia in Greece during the COVID-19 era as outlined in various studies presented in chronological order (n = 7)

Table 1b
Epidemiological aspects of candidaemia in Greece during the COVID-19 era as outlined in various studies presented in chronological order (n = 7) and the prior positive blood culture, were considered to represent new episodes.

Species identification and antifungal susceptibility testing
Candida BSI isolates were identified to species level by MALDI-ToF MS (Bruker Daltonics, Bremen, Germany).C. auris in vitro susceptibility was assessed using the Clinical and Laboratory Standards Institute (CLSI) M27-Ed4 [16] with the Centers for Disease Control and Prevention's (CDC) tentative resistance breakpoints [17] and previously proposed CLSI epidemiological cut-off values [18].Hence, C. auris isolates were classified as non-resistant/resistant to amphotericin B, echinocandins and fluconazole and wild type/non-wild type to the other drugs.For other Candida spp., the in vitro susceptibility profile was determined with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Definitive Document (E.DEF) 7.3.2[19] following the corresponding guidelines for interpreting the minimum inhibitory concentration (MIC).

Statistical analysis
The incidence of candidaemia was expressed as the ratio of Candida BSI episodes per 1,000 hospital and unit (ICU and non-ICU) admissions and per 10,000 hospital and unit (ICU and non-ICU) bed days.Antifungal consumption data were reported as defined daily doses (DDD) and expressed in DDD/100 hospital bed days using the antimicrobial consumption (AMC) tool 2019 version 1.9.0 (World Health Organization (WHO) Collaborating Centre for Drug Statistics Methodology, Oslo, Norway).Trend over time was evaluated by one-way ANOVA followed by post-test for linear trend.Changes in species distribution and in vitro susceptibility over the years were assessed statistically using Fisher's exact test.A p value of < 0.05 was considered to reveal a statistically significant difference.All data were analysed using the statistics software package GraphPad Prism 8.0 (GraphPad Software, San Diego, United States (US)).

Literature review
We retrieved seven articles whose extracted data are presented in   2).

Non-intensive care unit setting
As regards the non-ICU setting, the overall incidence of candidaemia increased significantly from 0.7 (range: 0.6-0.8)episodes per 1,000 hospital non-ICU admissions in the pre-COVID-19 era to 0.9 (range: 0.9-0.9)episodes per 1,000 hospital non-ICU admissions in the early COVID-19 era and further to 1.    [18] and EUCAST ECOFFs for the other Candida spp.were used (where available).

Antifungal susceptibility profile
Information on in vitro antifungal susceptibility was available for a subset of 336/394 (85%) isolates (Table 3 and Supplementary Table S1).All strains were nonresistant/wild type to amphotericin B and echinocandins except one C. auris isolate (pan-echinocandin-resistant harbouring an FKS1 S639F mutation with anidulafungin, caspofungin and micafungin CLSI MICs > 8 mg/L), which was recovered from a haematologic COVID-19 ICU patient who had experienced repeated exposure to anidulafungin.Among the azoles tested, reduced susceptibility to fluconazole was mostly seen.Namely, 1% of C. albicans, 84% of C. auris and 32% of C. parapsilosis SC strains were fluconazole-resistant, whereas 2% of C. parapsilosis SC isolates were categorised as susceptible at increased exposure.

Discussion
This study describes the epidemiological trends of Candida BSI in the patient population at Attikon University General Hospital of Athens, Greece in the wake of the COVID-19 pandemic.During the COVID-19 era (2020-2023), a significant rise in the incidence of candidaemia was observed in both ICU and non-ICU settings with most candidaemic patients admitted for reasons other than COVID-19.A shift from C. albicans to C. parapsilosis SC as the main aetiological agent of candidaemia was detected, along with an increased resistance rate to fluconazole for C. parapsilosis SC.Worryingly, C. auris-driven BSI and their transmission dynamics in the hospital environment have emerged in the transition to the post-pandemic era, raising the overall rate of fluconazole-resistant Candida BSI isolates to 41% at the end of the COVID-19 era in 2023.
The constantly evolving epidemiology of candidaemia globally has significant implications for patient management and highlights the need for continuous regional monitoring [12], specifically during extreme events such as the unexpected COVID-19 pandemic, which may have a considerable impact on the epidemiological pattern of healthcare-associated infections [2].To date, the contemporary prevalence and species distribution of candidaemia in Greece remains largely unknown and data on the antifungal resistance profile of the involved bloodstream isolates are lacking.During the COVID-19 era, the majority of studies on Candida BSI in Greece were carried out in a COVID-19 ICU setting during the early phase of the pandemic and included few patients.As a consequence, their results may not be broadly applicable to all candidaemic patients reflecting the current at that time local spectrum of the infection.Therefore, we aimed to provide an overview of the epidemiology of Candida BSI in the patient population at Attikon University General Hospital of Athens, encompassing the beginning of the pandemic and the WHO's declaration of the end of the COVID-19 emergency phase.
The incidence rate of candidaemia at Attikon has been stable over a 10-year period (2009-2018) [15].Nevertheless, a nearly threefold increase was noted during the pandemic.In total, we identified a higher number of candidaemic episodes in patients admitted to Attikon for reasons other than COVID-19 (249/365, 68%), as previously described (64-74%) [20][21][22].Two-to threefold differences have been also recorded in single-centre studies that compared the incidence of Candida BSI before and during the COVID-19 pandemic in Spain (1.4 vs 2.6 cases/10,000 patient days) [20] and Brazil (0.2 vs 0.6 cases/1,000 patient days) [22], while no data have been previously reported for Greek hospitals precluding country-wide comparisons.Such an increase could be potentially attributed to either the expansion of the number of patients prone to develop candidaemia [1] or the higher propensity for patient-to-patient transmission since the COVID-19 pandemic negatively impacted infection control measures.
The difference was more pronounced in the context of the ICU setting.The overall incidence of Candida BSI rose considerably (almost a fourfold increase in bed days) during the pandemic, with the infection rate being higher in COVID-19 patients, as previously described [20].Of note, several retrospective studies have shown a significantly increased incidence of candidaemia among COVID-19 patients requiring ICU care in comparison to historical non-COVID-19 cohorts [23,24], which is in agreement with our findings.Nevertheless, these data should be interpreted with caution given that the patient populations being compared are different in terms of pathology and underlying comorbidities.Moreover, our results are consistent with a previously published comparator study reporting that the incidence of Candida BSI increased also in the non-COVID-19 group during the pandemic [22].The difference observed may have been driven by the fact that hospital admissions for non-COVID-19 cases were restricted to severe aetiologies, i.e. patients who were more likely to have longer length of stay and higher risk for nosocomial infections, in conjunction with impaired infection control practices during the surge of the pandemic.
Regarding the local epidemiology, analogous trends have been previously recorded across two Greek adult ICUs.A single-centre study performed in Athens found an almost threefold increased incidence rate of candidaemia in COVID-19 patients as compared with historical non-COVID-19 controls (103.3vs 38.0-42.0/1,000ICU admissions) [8], which is lower than the fivefold increase observed at Attikon Hospital.On the other hand, a single-centre study carried out in Patras (south-western Greece) reported a nearly fourfold greater incidence density of ICU-acquired candidaemia during the pandemic era (4.5 vs 16.9/1,000 patient days), in line with our findings.Nevertheless, similar rates were recorded among COVID-19 and non-COVID-19 patients in the same time period (19.1 and 13.8/1,000 patient days, respectively) [13], as opposed to our study.Similar intra-country variations have been previously described [25], probably due to differences in the time period of the pandemic regarding the impact of its diverse transmission waves, local practices and surveillance protocols, the consumption of antifungal drugs and the patient populations taking into account the variable percentages of surgical/oncological cases.Amid the pandemic, C. parapsilosis SC outranked C. albicans as the dominant causative agent of candidaemia at Attikon Hospital (40% vs 37% in the pre-COVID-19 era) [15].This is in accordance with data derived from southern European countries (Italy and Spain) demonstrating a corresponding shift in favour of C. parapsilosis SC-driven BSI in the general patient population during the COVID-19 era [25,26].Overall, the proportion of other previously isolated non-albicans Candida spp.such as C. tropicalis, which increased in Brazil [22], the US [21] and Spain [25] in the same time period, remained stable at Attikon.No difference was observed in the dispersion of Candida bloodstream isolates between COVID-19 and non-COVID-19 ICUs during the pandemic, which is in line with worldwide trends [23,24].However, significant differences in species distribution outside of the ICU setting were noted, like the dominance of N. glabratus candidaemic episodes in non-COVID-19 patients, as previously described [27].A possible explanation would be that COVID-19 patients do not have the risk factors commonly linked to the development of N. glabratus invasive infections, particularly recent abdominal surgery and exposure to azoles.
Yet, our most concerning finding was the dissemination of C. auris candidaemias altering the in-hospital spectrum of the infection.The spread of   increase in the incidence of fluconazole-resistant C. parapsilosis SC during the COVID-19 era has not been driven by the selective pressure of fluconazole and voriconazole consumption, as previously described [33].The latter advocates that the main reason for the epidemiological changes described here might have been because of poor adherence of infection prevention and control measures.During the pandemic the following factors that could have contributed to the increase have been noticed: (i) extended wear/reuse of gloves by healthcare workers for the care of multiple patients, (ii) staff movements across different hospital units, (iii) previous hospitalisation in hospitals located in the Attica region where C. auris and C. parapsilosis outbreaks have been documented [34,35], (iv) systematic screening not being performed for all patients at admission and randomly during hospitalisation, (v) isolation/cohorting of patients colonised with C. auris until discharge not implemented due to space and nursing staff shortages.
It should be acknowledged that our study is limited by its retrospective nature, while its results may have been affected by local hospital-associated practices in a period of heavy workload and distinct patient populations.However, it contributes to the existing knowledge regarding the epidemiology of candidaemia in Greece, and particularly the extent of C. auris-driven infections, by providing up-to-date data.Hence, the findings presented herein may be an indicator of an emerging regional problem and can be considered as a point of reference paving the way for future epidemiological surveys.In addition, as similar trends have been reported in other countries, mainly in the south of Europe, the present report describes a situation that may lead to the end of the era of empiric azole therapy against Candida infections.

Conclusion
The incidence of Candida BSI increased worryingly during the COVID-19 era.C. auris emerged among the major causative agents, advocating the need for ongoing vigilance and strict adherence to infection prevention and control practices in healthcare settings, whereas further studies are required to shed light on its potential regional endemicity.Meanwhile, fluconazole resistance in C. parapsilosis SC exhibited a rising trend in our large tertiary hospital, which together with the rise of C. auris increase the rate of azole-resistant Candida isolates to 41% of all clinical isolates.Prompt implementation of surveillance and antifungal stewardship activities is crucial to contain the selection and spread of echinocandin resistance in C. auris.

Table 1 .
Overall, the study periods encompassed the early COVID-19 era (March 2020 to December 2021, 6/7 articles), while all articles reported data from single hospitals.

Table 3
In vitro susceptibility profile to nine antifungals of major Candida spp.bloodstream isolates from patients, Attikon University General Hospital of Athens, Greece, 2020-2023 [17]/ECOFFs: epidemiological cut-off values; I: intermediate; MIC: minimum inhibitory concentration; NA: not applicable; ND: not determined; Non-R: non-resistant; R: resistant; S: susceptible; SS: sensu stricto; WT: wild type.aCentersfor Disease Control and Prevention's tentative resistance breakpoints for C. auris[17]and European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints for the other Candida spp.were used (where available).b Clinical and Laboratory Standards Institute ECVs for C. auris